The objectives of this NRSA individual fellowship are to 1) facilitate the development of research skills that will allow the candidate to become an effective physician-scientist and 2) investigate the mechanisms behind oxidant induced endothelial Ca2+ influx and lung permeability in Acute Respiratory Distress Syndrome (ARDS). ARDS is a common, sometimes fatal lung condition characterized by increased permeability of the lung endothelium, allowing for fluid to enter the alveoli, resulting in an inability to oxygenae the blood. Reactive oxygen species (ROS) are oxygen derived molecules known to be increased in many diseases including ARDS, and also known to cause cellular stress and injury. In addition to ROS, abnormally high intracellular Ca2+ levels have been shown to lead to increased endothelial permeability. However, the relationship between ROS and Ca2+ entry into the lung endothelium remains unclear. The candidate has preliminary data that shows that Ca2+ levels in lung endothelial cells rise after they have been exposed to ROS, and that this rise in Ca2+ is absent in cells that are lacking a protein called CD36. CD36 is a receptor that normally binds fatty acids. It has been shown by others to participate in calcium and ROS signaling in immune and neuronal cells, but its role in lung endothelial cells has not been examined. The focus of this project is to examine how CD36 influences Ca2+ influx in response to ROS in the lung endothelium. The long-term goal of this proposal is to identify novel mechanisms of regulation of Ca2+ entry into endothelial cells that can be targeted by drugs designed to help patients in ARDS as well as other diseases where the endothelium is abnormally permeable. In Specific Aim 1, using fluorescent Ca2+ imaging and siRNA on lung endothelial cells, the candidate will test whether TRPV4, a Ca2+ channel shown in the candidate's preliminary studies to be possibly involved in ROS-induced Ca2+ entry, is the channel that is responsible for Ca2+ influx. In Specific Aim 2, using immunoblots and Ca2+ imaging on both normal endothelial cells and endothelial cells with genetic deletion of CD36, the effect of ROS on CD36 and another protein associated with CD36 called Fyn will be assessed. The goal of this aim will be to determine the pathway by which ROS eventually causes opening of Ca2+ channels. In Specific Aim 3, using a model of in vivo lung injury and measurement of in vivo Ca2+ in mice, the effect of drugs blocking various members of the proposed pathway linking ROS, CD36 and TRPV4 on calcium entry and development of lung edema will be tested. The goal of these experiments is to understand the mechanisms behind Ca2+ entry and subsequent loss of barrier function in lung endothelial cells injured by ROS.